Outer membrane proteins (OMPs) and lipoproteins (LPs) of Gram-negative bacteria are of enormous importance as virulence factors, stabilizers of outer membrane integrity, protective immune targets (vaccines), and proinflammatory agonists that elicit innate immune responses. LPs, in particular, also tend to be superior serological test reagents given that they induce strong antibody responses via their intrinsic adjuvant properties. Virtually nothing is known about the OMPs and LPs of Francisella tularensis strains that are pathogenic for humans (e.g., F. tularensis Schu4). The substantial biohazard issues surrounding the laboratory manipulation of such virulent strains undoubtedly have contributed to this dearth of information. Consequently, given the potential importance of OMPs and LPs as candidate vaccines and diagnostic reagents for tularemia, the Specific Aims of this proposal are: (1) To identify and characterize the major OMPs of F. tularensis strain Schu4 (Schu4); (2) To identify the LPs of Schu4, with emphasis on characterizing those within the outer membrane; (3) To clone and express in E. coli Schu4 0MP and LP genes as fusion proteins for the generation of specific polyclonal and/or monoclonal antibodies (antibodies will be used in membrane topology and surface localization studies); and (4) To perform vaccine studies in the murine model of tularemia using candidate Schu4 0MPs and LPs identified in Specific Aims 1-3. The studies will include state-of-the-art experimental approaches for OMP and LP identification, combined with a F. tularensis pulmonary challenge model for vaccine experiments in mice. Interdigitation with the other four complementary projects of this P01 Program will markedly enhance the success of this project. The combined studies could lead to the discovery of new diagnostic reagents and acellular vaccines for tularemia.